Kinesin and the unconventional myosins V and VI are molecular motors responsible for many types of subcellular movement. Mutations lead to diseases, yet fundamental questions remain about the mechanism of motion. For example, do these dimeric motors move in a "hand-over-hand" or "inchworm" mode? We have developed a single molecule fluorescence technique that can provide answers. We attach a fluorophore to the motor protein and determine its position with 1.5 nm spatial localization, 0.5 second time resolution, and a photostability that enables observations for several minutes. The method relies on analyzing the center of the emission point-spread-function (PSF), which we have shown represents the position of the fluorophore under appropriate conditions. The technique complements fluorescence resonance energy transfer, which is sensitive to the 2-10 nm range, and optical traps, which can (only) measure center-of-mass motion (under load) with nanometer precision. We will measure the step size, angular orientation, and relative positions of the head, neck and coiled-coiled stalk of these motor proteins during motility, under no-load and loaded conditions. More specifically, A) single molecule nanometer-localization of a single fluorophore on the head of motors will be able to differentiate inchworm from hand-over-hand models (8 nm and 16 nm predicted step size, respectively, for kinesin). Using this technique, we have recently shown Myosin V moves in a hand-over-hand mechanism (Yildiz et al, Science, 2003). B) We present initial results achieving single molecule nanometer resolution -i.e., measuring the distance between two dyes with nanometer precision- which we will use to map out the relative distance and motion of two parts within the motors. For example, by attaching two fluorophores that emit different colors, one on each head, the hand-over-hand model will lead to alternating PSFs; the inchworm model will lead to the PSF of one color always leading. C) Single-molecule orientational imaging will be developed and applied to detect angular changes in the head, neck and stalk regions. An inchworm model predicts no rotation of the stalk, whereas a symmetric hand-over-hand predicts a rotation, and an asymmetric hand-over-hand model may not have a rotation. D) Through statistical analyses of data via Hidden Markov Methods, a method originally used for single ion channel analysis, we will also learn if there are several sub-conformations corresponding to a particular position of the head. Experiments will use human ubiquitous kinesin, chicken brain myosin V, and porcine myosin VI - the latter two take approximately 36 nm center-of-mass steps. Application to rat brain cytoplasmic dynein, another motor, is briefly presented. We anticipate our techniques will be applicable to many protein and nucleic acid systems.